Extending Precise Spatiotemporal Control of Engineered Bacteria Through Acoustic Manipulation
Modular plasmid architecture for the rapid construction and characterization of temperature-controlled genetic circuits in E. coli. (a) Individual elements of genetic circuitry are cloned separately as parts, thus allowing for rapid assembly of genetic circuits with virtually any combination and orientation of these parts. (b) Optimized architecture for circuits regulated by the thermal bioswitch TlpA-42. Both the bioswitch and fluorescent reporter mScarlet-I are under the control of strong ribosomal binding sites (RBSs), and TlpA is driven by medium strength constitutive promoter (P18) while the m-Scarlet-I is under the control of PtlpA promoter. (c) Thermal switching performance at 37 vs 42ºC. Data provided by Gollihar lab.
In this project, we will extend the technologies allowing precise spatiotemporal manipulation of engineered bacteria from thermal actuation to direct physical manipulation using acoustic radiation force (ARF). ARF is based on ultrasound waves, which can be applied in a wide range of biological devices, materials and tissues to apply forces on objects with mechanical properties distinct from their surrounding medium. By engineering bacteria to express a unique class of air-filled proteins giving the cells reduced density and stiffness relative to water, we will enable their physical manipulation, sorting and patterning using sound waves. Unlike our successful previous work on thermal control, ARF allows direct physical manipulation, allowing cells to be moved and arranged in space. As a result, it will provide a unique capability complementary to our thermal control technology. Furthermore, because ARF relies on ultrasound, it will have much greater applicability and scalability in biological engineering than light-based techniques, which break down due to light scattering in biological media. Over the next two years, building on extensive preliminary data, we will demonstrate the use of ARF to quickly and inexpensively sort cells based on the output of their gene circuits and establish the ability to quickly and inexpensively pattern cells into intricate spatial patterns to facilitate the development of living materials. We will integrate these capabilities with our previously developed thermal bioswitches, completing a versatile platform for the biological engineering of cells and biomaterials.
